Cytoplasmic male sterility (csm) is the most useful maternally inherited trait of higher plants available to breeders. This trait provides a reliable, inexpensive method to emasculate a female parent plant for the production of hybrid seed and is therefore commercially important. Three major types of nuclear/cytoplasmic interactions give rise to cms in corn (Zea mays L.). cms C-, cms T- and cms S-types can be differentiated by nuclear genes that restore pollen fertility to cms-plants, e.g., a corn inbred strain C0150 has genes which restore fertility only to cms C-types; inbred strain NYD410 restores fertility only to cms T-types and MS64-7 restores fertility only to cms S-types. Fertility restoration in cms S was found to be mediated by a single gene locus Rf3, while T is restored by two gene loci, Rf1 and Rf2 (Duvick, D. N. (1965) Adv. Genet. 13: 1-56; Laughnan, J. R. and S. J. Gabay (1975) in Genetics and Biogenesis of Mitochondria and Chloroplasts, eds. Birky, W. W., Jr. Perlman, P. S. and T. J. Byers (Ohio State Univ. Press, Columbus, OH, pp. 330-349). The standard restorer gene (Rf) for cms-S has been mapped to the long arm of chromosome 2(Gabay-Laughnan, S. J. and J. R. Laughnan (1979) Maize Genet. Coop. Newsletter 53:92-93). Almost all cms corn plants that have been studied can be restored to fertility by one of the above three types.
In the past the three cms types have varied in their commercial significance. Because the cms T-type induced stable sterility (Table 1) in a large number of inbred strains, it was used to produce about 95% of hybrid corn in 1970.
It was, however, quickly abandoned because it was susceptible to race T of the fungus Helminthosporium maydis, which destroyed 15% of the U.S. corn crop in 1970. C-type cms sterilizes fewer inbreds than cms T-type but more than cms S-type, and some breeders have replaced cms T-type with cms C-type. The lesson learned from the H. maydis epidemic was that wide scale genetic uniformity in crop plants leads readily to selection of specific pathogens. It is therefore important to have a variety of stably sterile cms strains available.
Even though all cms-plants can be categorized into cms C-, cms T- and cms S-types, it is important to recognize that there are genetic differences within these types and these variations can be recognized by differences in restoration capacity when the genome of different inbred strains is associated with these male sterile cytoplasms.
LBN cytoplasmic male sterility is a sub-type of the cms S-type. cms S-type cytoplasmic sterility has been known since the 1950's but was not the subject of intensive research until recent years. When the H. maydis Race T epidemic of 1970 discredited cms T-cytoplasm, cms S-type did not provide a reasonable alternative because it did not provide stable sterility in association with a wide variety of corn inbreds. Several hundred cases of inherited cytoplasmic changes from cms S-male sterile to a male fertile condition have been identified (Singh, A. and J. R. Laughnan (1972) Genetics 71:607-620). Instead, producers of hybrid corn seed turned to hand detasseling, a mechanical form of emasculation, or to cms C-type, which sterilizes more inbreds than the cms S-type.
cms S-type plants possess two plasmids (S1 and S2) in the mitochondria. S1 has a length of 6.4 kbp and S2 is 5.4 kbp. These plasmids may be involved in the mechanism of cms S-type male sterility, because, in cms-S plants that spontaneously revert to fertility, the plasmids have apparently become integrated in the high molecular weight mitochondrial DNA (hmw mtDNA). S1 and S2 have terminal inverted repeats characteristic of transposable elements. The mitochondrial nucleic acids probably encode the maternally inherited component of cms, because, in corn, differences in cms C-, cms T- and cms S-types are more strongly correlated with differences in mitochondrial DNA (mtDNA) than with differences in chloroplast DNA. These two organelles, mitochondria and chloroplasts, contain maternally-inherited nucleic acids of plants. Leaver et al. (Leaver, C. J. and M. W. Gray (1982) Ann. Rev. Plant Physiol.) found that in the cms-T system, a mitochondrially-encoded protein unique to cms-T is changed when nuclei containing cms-T restorer genes are introduced. These observations support the hypothesis that restoration of fertility in cms plants results from interaction between mitochondrial and nuclear gene products.
There is a strong correlation between the S1 and S2 DNA's and cms-S (Levings, S. et al (1980) Science 209:1021-1023). The mtDNA of seven cms-S revertants to fertility were analysed. It was found that the S1 and S2 plasmids had virtually disappeared in all seven revertant strains and this disappearance was associated with the presence of new fragments in restriction endonuclease digests of the hmw mtDNA. Labelled probes of S2 DNA hybridized to some of these new bands. Further evidence suggesting that the S1-plasmid is important in reversions to fertility was the quantitative decrease of S2 in M825 (the sweet corn inbred that produced the highest reversion rate). S1 and S2 plasmids usually occur in equimolar quantities in cms-S mitochondria but in the M825 nuclear association, the S2 is decreased in amount. The implication was that the fertility of the revertants was caused by integration of S2 or by the mtDNA rearrangements resulting from the integration and that cms-S was related to the non-integrated state of the plasmids. More recent studies have shown that sequences homologous to those of S1 and S2 plasmids are integrated in the hmw mtDNA of other non-sterile ("normal") cytoplasms thus supporting the idea that integration of S1 and S2 is correlated with fertility.
However, the role of S1 and S2 is not clear. S1 and S2 hybridize to the hmw mtDNA of S-sterile as well as that of normal and revertant cytoplasms. If plasmid integration into hmw mtDNA does result in fertility, then an analogy to transposable element systems might explain the presence of S1 and S2 sequences in hmw mtDNA of S-sterile plants (Levings, C. S. III, et al. (1980) Science 209:1021-1023). Certain transposable elements duplicate themselves in the process of excising, leaving the duplicate copy in the original site (Shapiro, J. A. (1979) Proc. Nat. Acad. Sci. USA 76:1933-1937). Levings, et al. (supra) concluded that "the presence in the mitochondrial DNA of sequences homologous to the S1 and S2 DNA's after their excisions would be anticipated if excision events behave similarly [to the prokaryotic system]". But the question of whether the integrated or non-integrated state of the plasmids has anything to do with male sterility has not been settled.
Another report (Weissinger, A. K., et al. (1982) Proc. Nat. Acad. Sci. USA 79:1-53) identified two other plasmids, R1 and R2, in non-sterile cytoplasms from 12 races of South American maize. R2 appears to be identical to S2 in size and restriction sites. R1 contains most of S1 plus 1000 additional base pairs. R1 and R2, like S1 and S2, have terminal inverted repeats of approximately 150 base pairs. The hmw mtDNA of the R-containing cytoplasms was markedly different from the hmw mtDNA of cms-S, as shown by the patterns of restriction digests.
Other low molecular-weight mtDNA's (lmw mtDNA) in addition to S1 and S2 occur in "normal" and the three cms cytoplasms: C-, T- and S- cytoplasm (Kemble, R. J. and J. R. Bedbrook (1980) Nature 284:565-566). One of these lmw mtDNA's, which is found in all cytoplasms, exists in linear (L), open circular (OC) and supercoiled (CCC) forms. Another, named "T" (36, 51) is 2.3 kbp long in N-, cms C- and cms S- cytoplasms and 2.0 kbp long in cms T-cytoplasm. Koncz, -et al. [(1981) Mol. Gen. Genet. 183:449-458] reported that S2 probes hybridize strongly to these 2.0 - 2.3 kbp DNA's. Finally, C-cytoplasm alone has two additional circular DNA's (called "C") approximately 1600 and 1400 bp long. The role, if any, of these various lmw mt DNA's in the cms phenomenon is unknown.
To summarize, the best evidence for the involvement of the S-plasmids in cms comes from the analysis of seven revertants to non-sterile cytoplasm. In these seven cytoplasms, the S1 and S2 DNA's disappeared, new bands appeared in restriction digests of the hmw mtDNA and S2 probes hybridized to some of these new bands. Nevertheless, the precise relation (if any) of S1 and S2 to cms-S or to the reversion to fertility is unclear.
The main evidence supporting the correlation between the S-bands and cms-S (Kemble, R. J. et al. (1980) Genetics 95:451-458; Forde, et al. (1980) Genetics 95:443-450) involved the analysis of a large number of cms cytoplasms which mostly originated from the Cornell collection. All cms strains that had been identified as cms-S by the restoration gene pattern contained the S1 and S2 bands and also had a characteristic set of mtDNA in vitro translation products. B and D, which are male-sterile cytoplasms previously left unclassified (Beckett, J. B. (1971) Crop. Sci. 11:724-727; Gracen, V. E. and C. O. Grogan (1974) Agron. J. 65:654-657) because of unusual restoration patterns, were also found to have the S-bands and the in vitro translation products characteristic of S. All other cytoplasms could be classified as cms T-, cms C-, "normal" or EP (from teosinte).
Another characteristic that distinguishes cms S from cms T and cms C is gametophytic restoration (Buchert, J. G. (1961) Proc. Nat. Acad. Sci. USA 47:1436-1440). In a gametophytic system, plants are restored to fertility at the level of the gametophyte, i.e., the pollen grain. When a cms-S plant heterozygous for nuclear restorer genes produces pollen, then only pollen containing the restorer (Rf) allele develops normally. Pollen in the same tassel containing the non-restoring (rf) allele aborts. In contrast, fertility restoration of cms-C and cms-T is sporophytic. All pollen of Rf rf plants develops normally in a sporophytic system, even though half of the pollen does not carry the Rf allele. Thus the pollen viability is determined by the nuclear genotype of the mother plant, i.e., the sporophyte.
There are thus four criteria to distinguish cms-S: (1) presence of S1 and S2; (2) in vitro translation products of mtDNA; (3) gametophytic restoration; and (4) restriction digest patterns of mtDNA.